1. Field of the Invention
The present invention provides methods of diagnosis and prognosis of human chronic lymphocytic leukemia (CLL) in a subject a patient in need thereof. The methods of the present invention involve measuring the expression profile of two known genes: LPL and ADAM29; and determining the ratio of their expression to diagnose the presence of CLL or to prognose the likelihood of developing CLL, an aggressive or a stable form of the disease or the symptoms consistent with CLL.
2. Discussion of the Background
Chronic lymphocytic leukemia (CLL) displays a variable outcome. The classical Rai1 or Binet2 staging systems have allocated CLL cases into three major risk groups (low [stage 0 in Rai's and stage A in Binet's classification system], intermediate [stages I and II in Rai's and stage B in Binet's classification system], and high [stage III and IV in Rai's and stage C in Binet's classification system]), according to tumor burden and the presence of anemia and thrombocytopenia. These staging systems have provided a basis for therapeutic stratification. Asymptomatic patients with a low tumor burden (Binet stage A) do not benefit from treatment with chlorambucil. However, the disease in half of these patients will progress and both staging systems fail to initially identify such patients. The advent of new treatments such as purine analogues and monoclonal antibodies directed against CD20 and CD52 are able to induce complete remissions and may allow early treatment for asymptomatic patients whose disease is likely to progress.3 Accurate identification of these patients is therefore increasingly important.
Serologic markers such as lactic dehydrogenase, beta2-microglobulin4, soluble CD235 and thymidine kinase4,6 are essentially indicators of disease activity and/or load, although some can anticipate disease progression7. Phenotypic expression of CD38 has been associated with aggressive disease8, but the threshold level for positive cases, if it exists at all, remains a matter of debate.9-11 Genomic aberrations correlate well with either good (isolated 13q−) or poor (17p−; 11p−) prognosis in CLL10,12,13, though their occurrence as a second malignant hit cannot be definitely excluded.
The mutational status of immunoglobulin heavy chain variable (IgVH) genes has been considered as the best prognostic marker in CLL. In an initial study, the present inventors observed that at least half of CLL cases carried mutations using a cut-off of 98% germline homology.14 This was further confirmed by others,15 some of which also correlated the IgVH mutational status to clinical behavior.8,16,17 Mutated (MT) patients usually demonstrate a favorable evolution when compared to unmutated (UM) cases (≧98% germline homology), which are characterized by progressive disease, continuing treatment needs and a high proportion of CLL-related deaths. This analysis remains costly, time-consuming and inaccessible for most medical facilities. Consequently, the detection of appropriate, reliable surrogate markers for IgVH mutational status has retained worldwide attention.
CD38 was the first candidate proposed to replace IgVH sequencing,8 with positive and negative cases corresponding respectively to UM and MT patients, but finally demonstrated insufficient specificity (about 30% discordance for each group).11 In addition, its expression can vary during the course of disease.11 Surprisingly, recent reports indicated that ZAP-70 mRNA, normally expressed in T and NK lymphocytes, is also transcribed in CLL B-cells lacking IgVH mutations.18,19 Two further series have confirmed these findings at the protein level,20,21 suggesting a pivotal role for ZAP-70 in prediction of IgVH mutational status. There is however some controversy to whether ZAP-70 is really a good surrogate marker since two recent reports failed to demonstrate a significant concordance between its expression and the degree of somatic mutation in the IgVH genes.22,23 
Accordingly, there remains a critical need for a safe, inexpensive and accurate means to diagnose CLL and prognostic methods involving the same.
To address this need, in a study of gene expression profiling performed on 18 CLL cases, the present inventors identified a limited set of genes (n=85), which were expressed differentially between progressive UM and stable MT CLLs (Vasconcelos, et al., manuscript submitted July 2004). These results were validated by real-time quantitative polymerase chain reaction (RQ-PCR) for 18 genes on the same cDNAs that were hybridized on the DNA chips. From these RQ-PCR experiments, 4 genes in addition to ZAP-70 appeared to provide a better segregation of the 2 groups of CLLs. They included the lipoprotein lipase (LPL) and spartin (SPG20) genes, whose expression was higher in UM patients, while a disintegrin and metalloproteinase 29 (ADAM29) and nuclear receptor-interacting protein 1 (NRIPI) genes were found at much higher levels in MT cases. These findings led the present inventors to investigate, in an independent and larger CLL series, which of these 4 genes (isolated or in combination) could represent the best surrogate marker for IgVH mutational status, and how they would compare to ZAP-70 protein expression.